Loss of Function variants and Transcription Manuel Rivas, University of Oxford Geuvadis RNA Sequencing Analysis Meeting 15/04/2012

Loss of Function variants (Putative) LoF variants: Splice site variants Nonsense and Readthrough mutations Frameshift indels. Exon Deletion and Duplication Gene Deletion and Duplication Gene Fusion* 5’ AG GT[AG]AG AG G 3’

What can we learn about LoF using RNA sequencing data Effect of splice variants on transcripts Exon Skipping Intron Retention mRNA Surveillance Pathways Escape NMD Trigger NMD What are the sequence properties that trigger NMD pathway or dictate escaping NMD pathway. 3. NMD efficiency in individuals. Do rates vary between individuals? CNVs Gene Fusion – impact of gene fusions in transcription? Do they properly get transcribed? Exon/Gene Deletions and Duplications – impact of exon/gene deletions and duplications in transcription?

Splice Site Variants ` ` ` ` Initial analysis of Exon Quantification Data using splice variant annotation successfully recapitulates splicing event. (IFIH1 Exon Skipping c.IVS14+1G>A) ` G A ` ` ` Annotation -> Evaluate Empirical Effect on Transcript

mRNA surveillance – Nonsense Mediated Decay (NMD) IFIH1 Transcript Escapes Nonsense Mediated Decay. Model 1: NMD is not triggered Model 2: NMD is triggered ` G A ` ` ` Propose Statistic that detects Nonsense Mediated Decay EXAMPLE 1: ESCAPE NMD

mRNA surveillance – Nonsense Mediated Decay (NMD) CIAO1 premature stop variant triggers Nonsense Mediated Decay. Model 1: NMD is not triggered Model 2: NMD is triggered Propose Statistic that detects Nonsense Mediated Decay EXAMPLE 2: TRIGGER NMD REDUCTION OBSERVED THROUGHOUT TRANSCRIPT

Loss of Function variants and ASE Added Annotation Layer to VAT output from annotation program co-developed at Oxford. Fraction of LoF observations with allele specific expression detected. Crude measure at the moment based on Tuuli’s first run (n = 180 samples). Determines a two-sided confidence interval from the beta posterior.

Loss of Function variants and eQTL Analysis Dependent on frequency range. ASE most powerful for rare variants. eQTL suitable for common variants.

Methods and Analytical Strategies for LoF variants Splicing Effects based on Annotation Exon Skipping Intron Retention mRNA Surveillance Pathways Escape NMD Trigger NMD CNVs Gene Fusion Exon/Gene Deletions and Duplications

Splice Site Variants (Putative) LoF variants: Splice site variants Nonsense and Readthrough mutations Frameshift indels. Exon Deletion and Duplication Gene Deletion and Duplication Gene Fusions* Identify additional splice variants based on transcript assembly M. Sammeth and other groups.